Receptor-mediated endocytosis (RME) is a system for the transfer into the cell of large molecules involved in cell nutrition, activation or mitogenesis. Of the large number of receptor systems described, e.g. LDL receptor, epidermal growth factor, insulin receptor, transferrin receptor, none is understood in detail at the molecular level. This research proposal investigates this area of cell biology using as a model system the asialoglycoprotein receptor (ASGP-R) in a human hepatoma-derived cell line. The ASGP-R is well suited to these studies as there are purified ligands, anti-receptor antibodies, and purified receptor protein and fragments thereof available in the sponsor's laboratory. In addition the basic cell biology of the receptor protein has been well characterized by the sponsor over the last four years. Specific aims of this proposal include the study of phosphorylation of the ASGP receptor in intact cells and in isolated plasma membranes. The precise molecular domains of phosphate additions and a characterization of the kinase enzymes responsible for these reactions will be investigated in an in vitro system. Altered states of receptor phosphorylation will be investigated using biochemical agents that perturb cell function (ligand, phorbol esters, calcium ionophores). Preliminary data have been accumulated suggesting changes in receptor phosphorylation under these conditions as well as simultaneous changes in receptor phosphorylation and RME in these cells. These studies involve binding of radioactively labelled ligand, cell fractionation and immunological isolation of internalized receptor, and colloidal gold immunocytoelectron microscopy. The goal of these studies is to equip the candidate with a broad range of biochemical and morphological skills in cell biology, while investigating a basic problem in receptor biology in a well-characterized model system. This program will prepare the candidate for a subsequent application of this information to the regulation of hematopoietic progenitors by growth factor receptors. Techniques for the isolation and purification of these cells are well developed within the candidate's division. This will permit, when combined with the requisite biochemical tools, detailed studies on a molecular level into the pathophysiology of bone marrow failure syndromes, both congenital and acquired.